Embryonic stem cells (ESCs) are capable of unlimited self-renewal and retain the pluripotency to differentiate into all cell lineages in the body. Since ESCs undergo robust cellular proliferation and DNA damage occurs during normal cellular proliferation, it is critical for ESCs to possess stringent mechanisms to maintain genetic stability to prevent the passage of DNA damage to the progeny. Consistent with this notion, the spontaneous mutation rate is much lower in ESCs than in somatic cells. The mechanism to maintain genetic stability in ESCs remains unclear. I hypothesize that one primary mechanism to rid of DNA damaged-ESCs is to induce their differentiation into other cell types in order to maintain the genetic integrity of the self-renewing pool of ESCs. Recent discovery of the homeodomain protein Nanog as the master regulator of self-renewal allows us test this hypothesis. We hypothesized that the expression and activities of Nanog might be suppressed in response to DNA damage. In support of this notion, our recent findings indicate that DNA damage induces the differentiation of ESCs by suppressing the Nanog expression. Tumor suppressor p53 is required for this process and suppresses the expression of Nanog by direct binding to its promoter. I propose three aims to further elucidate the regulation of the expression and activity of Nanog after DNA damage and during ESC differentiation, and to study the potential roles of Nanog in cancers. Aim 1. To elucidate physiological importance of p53 family members-dependent downregulation of Nanog in mouse and human ESCs. Aim 2. Regulation of Nanog activities through protein-protein interaction in ESCs after DNA damage. Aim 3. To study the roles of Nanog in development, genetic stability and tumorigenesis. These studies have direct relevance to human health. In this context, the studies of the potential role of Nanog in tumorigenesis will help to develop new therapy for Nanog-overexpressing cancers, including germ cell tumors and breast carcinomas. In addition, these studies will shed light on mechanism that maintains genetic stability in human ESCs, which have a great potential for future human cell/tissue replacement therapy.